Imaging Biological Samples with ZEISS LSM 800 and LSM 880

A new reference list details scientific work with ZEISS LSM 8 family of confocal microscopes with Airyscan detector

The Confocal Laser Scanning Microscope (LSM) has becomeone of the most popular instruments for fluorescence imaging in biomedical research, because it affords researchers images with high contrast and a versatile optical sectioning capability to investigate three dimensional biological structures. The optical sectioning ability of an LSM is a product of scanning a focused laser spot, across a sample to create an image one point at a time. The generated fluorescence from each point is collected by the imaging objective and results from fluorophores in the sample that reside both in the desired plane of focus and in out of focus planes. In order to segregate the fluorescence emitted from the desired focal plane, an aperture (pinhole) is positioned in the light path to block all out of focus light from reaching the detector (traditionally a PMT). The traditional principle of the LSM beampath forces the user to compromise either on resolution or sensitivity. Resolution is increased by closing the pinhole, at the same time limiting the amount of light that is allowed to pass through to the detector. The Airyscan detector of LSM 880 and LSM 800 overcomes this challenge. The detector consists of 32 GaAsP PMT detector elements, which are arranged in a hexagonal array (Figure 1), positioned at a conjugated focal plane in the beam path the detector is functioning as the traditional LSM pinhole. This design makes it possible to collect more light (equivalent to a pinhole opened to 1.25 AU), whilst at the same time dramatically enhancing the resolution, with every detector element acting as an efficient pinhole with a diameter of only 0.2 Airy Unit (AU).

Instead of facing an either / or decision, a simultaneous enhancement of resolution by the factor of 1.7× and signal-to-noise by 4 – 8× was introduced to LSM imaging. Detailed descriptions of the theory and technology of Airyscanning can be found in separate technology notes. As an area detector, Airyscan can capture spatial information that is utilized to parallelize the scanning process, collecting 4 image lines simultaneously in the Fast mode (Figure 2). This means enhancing acquisition speed by a factor of 4 while keeping high pixel dwell times to efficiently collect emitted photons. In standard mode, the focused laser beam is moved along the x-axis to acquire one image line, before it is moved in the y-axis to acquire the consecutive image line. In Fast mode imaging, four image lines are acquired at the same time when moving the laser in the x-direction. A new publication list assembles some of the scientific work that has been done with LSM 880 and 800 systems. The great variety of applications collectively profits from the light efficient beam path of the LSM systems, and the unique combination of superresolution, high sensitivity and high speed imaging provided by Airyscan.